JP5787246B2 - Wound healing and pressure ulcer (bed sores) treatment, including keratinocyte migration / proliferation promoter - Google Patents

Description

  The present invention, based on gamma-glutamyltranspeptidase (GGT) inhibitory activity, promotes protein production in skin fibroblasts and promotes the migration and / or proliferation of keratinocytes in the skin The present invention relates to a protein production promoter for dermal fibroblasts and a keratinocyte migration / proliferation promoter, which contains a component having an action to act.

  The skin is divided into the epidermis (upper layer) and the dermis (lower layer). The epidermis forms the outermost layer of the skin, prevents the transpiration of moisture from the skin, and plays a role in protecting the living body from physical and chemical stimuli from the outside world. The keratinocytes constituting the epidermis (also referred to as “keratinocytes”) are present in the lowermost layer of the epidermis. The keratinocytes move to the outside (upper layer) of the epidermis while repeating cell proliferation in the basement membrane that forms the boundary with the dermis, and finally fall off as the horny layer in the uppermost layer of the epidermis. The keratinocytes form a dense epidermis while repeating this cycle over a month.

  In contrast, the dermis is a thick layer composed mainly of fibroblasts, fibrous tissue, and elastic tissue. Fibroblasts present in the dermis (referred to as “skin-derived fibroblasts” or “skin fibroblasts”) produce fibrous and hydrophilic proteins such as collagen and elastin, and these proteins enter the skin. By giving elasticity and strength, the skin can be kept fresh and supple.

  Skin fibroblasts also produce heat shock protein 47 (HSP47). HSP47 plays a role in helping collagen fibers produced by dermal fibroblasts form a triple helix and become mature collagen.

  However, the above-mentioned functions of the epidermis and dermis may be reduced due to the influence of external factors such as ultraviolet irradiation and drying, or aging, and as a result, the skin condition may be impaired.

  For example, if the keratinocyte migration / proliferation capacity decreases due to aging, etc., the turnover of cells constituting the epidermis is delayed, and as a result, the metabolism of the skin is accelerated, the barrier function of the skin is decreased, wrinkles, rough skin Symptoms such as etc. come to appear.

  In addition, when the production of collagen and elastin in skin fibroblasts decreases due to the influence of external factors such as UV irradiation and drying, or due to aging, the skin barrier function decreases and the moisturizing function and elasticity are reduced. As a result, skin tension and gloss are lost, and wrinkles, sagging, and other symptoms occur.

  Occurrence of wrinkles, sagging, etc. seen in the aging change of the skin is a major aging phenomenon in appearance and is a serious problem for many middle-aged and elderly people. Conventionally, as a method for improving such a skin symptom, it has been a mainstream to directly apply a cosmetic containing collagen and its degradation product peptide to the skin. However, since such cosmetics temporarily supplement the moisture retention of the skin surface, they did not exhibit a sufficient effect of improving skin symptoms.

  Under such circumstances, the development of cosmetics that increase the amount of collagen in the skin is aimed at preventing skin aging and / or improving skin symptoms, including maintaining skin tension and gloss, and improving skin moisturizing function. It has been advanced. For example, it is reported that an extract derived from a natural product has a type I collagen production promoting action (Patent Documents 1 and 2), and ascorbic acid is used as a component having an action of stimulating collagen synthesis constituting a dermal extracellular matrix. Have been reported (Patent Document 3). Further, the present inventors have found that the amount of collagen produced by dermal fibroblasts increases in association with a transient and weak decrease in the amount of glutathione in the dermal fibroblasts. Has reported a group of compounds having an activity to enhance collagen production (Patent Document 4). In addition, a commercially available peptide derivative (Matrixyl (registered trademark) or Matrixyl 3000) having a large market share as an anti-aging active ingredient such as reducing wrinkles has been reported to enhance collagen production in dermal fibroblasts ( Patent Documents 5 to 8). Matrixyl (registered trademark) is palmitoyl-pentapeptide 3 and Matrixyl 3000 is a mixture of palmitoyl oligopeptide (Pal-GHK) and palmitoyl tetrapeptide-7 (Pal-GQPR).

  On the other hand, GGT is known as an enzyme that degrades intracellular glutathione. GGT plays an important role as an enzyme that degrades glutathione, which is a pool of major cysteine (Cys) outside the cell, and supplies Cys necessary for glutathione biosynthesis to the cell. It is thought that there is a physiological effect that controls the amount of glutathione. The present inventors have reported GGT inhibitors having high activity and high selectivity in Patent Document 9 and Non-Patent Document 1.

JP 2007-186471 A (published July 26, 2007) JP 2007-302607 A (released on November 22, 2007) Japanese Unexamined Patent Application Publication No. 2004-075646 (published on March 11, 2004) JP 2006-151860 A (published June 15, 2006) International Publication No. 00/43417 (published July 27, 2000) Special Table 2002-524487 Publication (Announced August 6, 2002) US Pat. No. 6,492,326 (published on Dec. 10, 2002) US Patent Application Publication No. 2004/0132667 (published July 8, 2004) International Publication No. 2007/066695 Pamphlet (released on June 14, 2007)

Han et al., Biochemistry 46, 1432-1447 (2007)

  The various substances and skin compositions for improving skin symptoms disclosed in Patent Documents 1 to 4 are all focused only on enhancing the production of collagen, which is one of the constituent components of the dermis.

  In addition, the effect of commercially available Matrixyl (registered trademark) or Matrixyl 3000 is limited to the promotion of collagen production, and no effect on the production of other proteins having an anti-aging effect or the proliferation of keratinocytes has been reported.

  As described above, the dermis is composed of a plurality of proteins and cells including not only collagen but also elastin and the like. Furthermore, the skin condition is maintained by the epidermis and dermis both maintaining their structure and function. Therefore, in order to prevent skin aging or improve skin symptoms including maintenance of skin tension and gloss, improvement of skin moisturizing function, etc., functions to enhance production of multiple proteins other than collagen and skin There is a need for further development of a drug that has a function of enhancing cell proliferation and the like and can additively (synergistically) provide an effect of preventing skin aging or improving skin symptoms.

  The present invention has been made in view of the above-mentioned conventional problems, and its purpose is to promote the production of a plurality of proteins effective in preventing skin aging and improving skin symptoms, and migration of keratinocytes. Another object of the present invention is to provide a drug containing a compound having an action of promoting proliferation as an active ingredient.

  The inventors of the present invention have made various studies with the main purpose of solving the above-mentioned problems. As a result, the selective GGT inhibitor has (i) elastin production amount of dermal fibroblasts, HSP47 production amount, α -Increase smooth muscle actin (α-SMA) and mature collagen production, (ii) be able to activate skin fibroblasts, and (iii) promote keratinocyte migration and / or proliferation The present invention has been completed by discovering that it has new functions such as obtaining.

  That is, the protein production promoter for dermal fibroblasts of the present invention comprises a compound that inhibits γ-glutamyl transpeptidase as an active ingredient. Moreover, the protein production promoter for dermal fibroblasts of the present invention promotes the production of one or more proteins selected from the group consisting of elastin, HSP47, α-SMA and mature collagen.

  The keratinocyte migration / proliferation promoter of the present invention comprises a compound that inhibits γ-glutamyltranspeptidase as an active ingredient.

  In the skin fibroblast protein production promoter or keratinocyte migration / proliferation promoter of the present invention, the compound inhibiting γ-glutamyl transpeptidase preferably contains a phosphonic acid diester derivative having a leaving group.

  The dermal fibroblast protein production promoter or keratinocyte migration / proliferation promoter of the present invention comprises a compound that inhibits GGT (GGT inhibitor) as an active ingredient.

  This GGT inhibitor contains elastin that affects skin tension and freshness in skin fibroblasts, HSP47 that helps collagen maturation, α-SMA that is an indicator of skin fibroblast activation, and mature collagen Protein production can be enhanced simultaneously. In addition, migration and / or proliferation of keratinocytes that form the epidermis can be promoted.

  Therefore, if the protein production promoter or keratinocyte migration / proliferation promoter of the dermal fibroblast of the present invention is used, the amount of elastin, HSP47, α-SMA and mature collagen in the dermis is increased and the skin is increased. In addition to enhancing the tension and elasticity of the skin, it promotes the turnover of the epidermis, promotes the metabolism of the skin, and maintains and improves the barrier function of the skin. As a result, the skin is additively (synergistically) Can provide an anti-aging effect or an improvement effect on skin symptoms.

It is drawing which shows the measurement result of the intracellular elastin amount of the human skin origin fibroblast obtained in the Example, (a) is a figure showing the result of a Western blot, (b) is a figure of the band of a Western blot. It is a graph which shows the result of having analyzed the density quantitatively. BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows the measurement result of the intracellular alpha-SMA amount of the human skin origin fibroblast obtained in the Example, (a) is a figure showing the result of a Western blot, (b) is a figure of a Western blot. It is a graph which shows the result of having analyzed the density of the band quantitatively. It is drawing which shows the measurement result of the amount of intracellular HSP47 of the human skin origin fibroblast obtained in the Example, (a) is a figure showing the result of a Western blot, (b) is a figure of the band of a Western blot. It is a graph which shows the result of having analyzed the density quantitatively. It is a figure which shows the influence of the GGT inhibitor on promotion of the migration and proliferation ability of the human keratinocyte obtained in the Example. BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows the measurement result of the amount of intracellular mature collagen of the human skin origin fibroblast obtained in the Example, (a) is a figure showing the result of a Western blot, (b) is a band of a Western blot. It is a graph which shows the result of having analyzed the darkness of the quantity quantitatively.

  Hereinafter, an example of an embodiment of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range means “A or more and B or less”.

  The skin fibroblast protein production promoter of the present invention and the keratinocyte migration / proliferation promoter of the present invention contain a compound that inhibits GGT (GGT inhibitor) as an active ingredient. In the present specification, the “skin fibroblast protein production promoter” is intended to mean “a composition for promoting protein production in skin fibroblasts”. Further, in the present specification, the “keratinocyte migration / proliferation promoter” intends “a composition that promotes migration and / or proliferation of keratinocytes”.

  The protein production promoter for dermal fibroblasts of the present invention can significantly promote the production of elastin protein, HSP47 protein, α-SMA protein and mature collagen protein in dermal fibroblasts. Therefore, the “skin fibroblast protein production promoter” of the present invention includes “skin fibroblast elastin production promoter”, “skin fibroblast HSP47 production promoter”, “skin fibroblast α- It can also be read as “SMA production promoter” or “mature collagen production promoter for dermal fibroblasts”.

  In the present specification, the above-mentioned “elastin production promoter for dermal fibroblasts” intends “a composition for promoting the production of elastin in dermal fibroblasts”. Moreover, the above-mentioned “HSP47 production promoter for dermal fibroblasts” intends “a composition that promotes the production of HSP47 in dermal fibroblasts”. The “skin fibroblast α-SMA production promoter” is intended to mean “a composition that promotes α-SMA production in skin fibroblasts”. In addition, the above-mentioned “mature collagen production promoter for dermal fibroblasts” intends “a composition that promotes the production of mature collagen in dermal fibroblasts”.

  Here, the above-mentioned “mature collagen” refers to a collagen protein having a molecular weight of 70 to 90 kDa in which precursor collagen is aggregated and further crosslinked in and out of the molecule. “Collagen maturation” refers to the precursor collagen being cross-linked.

  In other words, “promoting the production of mature collagen in dermal fibroblasts” can be said to “promote the maturation of collagen in dermal fibroblasts”. Therefore, the above-mentioned “mature collagen production promoter for skin fibroblasts” can be rephrased as “collagen maturation promoter for skin fibroblasts”.

  In the present invention, “promoting the production of protein in skin fibroblasts” means a control skin fibroblast to which the protein production promoter of skin fibroblasts of the present invention is not applied, when measured simultaneously by the same method. This means that the amount of protein produced in skin fibroblasts to which the protein production promoter for skin fibroblasts of the present invention is applied is increased by 1.1 times or more compared to cells. In the present invention, “promote production of elastin in dermal fibroblasts”, “promote production of HSP47 in dermal fibroblasts”, “promote production of α-SMA in dermal fibroblasts”, and “ “Promoting the production of mature collagen in dermal fibroblasts” has the same meaning. As a method for measuring the amount of protein in skin fibroblasts, a conventionally known method can be suitably used. As shown in the Examples described later, for example, the amount of protein in skin fibroblasts can be measured by Western blotting.

  As shown in the Examples described later, the GGT inhibitor is a protein of HSP47 that has a function of helping to produce elastin protein and a function of maintaining collagen maturation alone in skin fibroblasts. Production, protein production of α-SMA, which is an index of dermal fibroblast activation, and protein production of mature collagen can be enhanced. As a result, it exerts an effective action in preventing and improving skin aging, specifically, preventing and improving wrinkles and sagging by improving skin tension and gloss, or maintaining and improving the moisture retention function of the skin.

  Furthermore, the GGT inhibitor has an action of promoting the migration and / or proliferation of cutaneous keratinocytes, which can also promote the migration and / or proliferation of cutaneous keratinocytes. Thereby, it has the effect | action which accelerates | stimulates the migration and / or proliferation of a skin keratinocyte markedly, and exhibits an effective effect | action in the promotion of the metabolism of skin, the maintenance improvement of the barrier function of skin, etc.

  In the present invention, “promoting keratinocyte migration and / or proliferation” means a control keratinocyte to which the keratinocyte migration / proliferation promoter of the present invention is not applied, when measured simultaneously by the same method. This means that the keratinocyte migration / proliferation ability to which the keratinocyte migration / proliferation promoter of the present invention is applied is improved by 1.1 times or more. As a method for measuring the migration / proliferation ability of keratinocytes, a conventionally known method can be suitably used. As shown in the examples described later, for example, keratinocytes that have been cultured until they reach a confluent state are scraped in a straight line using a pipette tip, and the extent of repair of the keratinocytes is measured by measuring the degree of repair of the scratch site.・ Proliferative ability can be measured.

  That is, the GGT inhibitor alone can enhance skin tension, elasticity, moisturizing function, etc., promote epidermal turnover to promote skin metabolism, and maintain and improve skin barrier function. it can. Therefore, if the protein production promoter for skin fibroblasts of the present invention containing the GGT inhibitor as an active ingredient and the keratinocyte migration / proliferation promoter of the present invention are used, they are additively (synergistically). In addition, it can bring about an anti-aging effect on skin or an improvement effect on skin symptoms.

  Hereinafter, an example of a specific embodiment of the present invention will be described.

  As the GGT inhibitor used in the present invention, a phosphonic acid diester derivative group (Patent Document 9) characterized by high enzyme selectivity and irreversible inhibitory activity can also be used. This group of phosphonic acid diester derivatives can be preferably used because of its particularly low biotoxicity.

  Specifically, for example, the following phosphonic acid diester derivative groups can be suitably used.

  1. General formula (1)

A phosphonic acid diester derivative represented by the formula (wherein at least one of R1 and R2 represents a leaving group):

2. General formula (1)
(In the formula, at least one of R1 and R2 is represented by the general formula (2) to the general formula (6).

(In the formula, R3 is any of an aryl group optionally having substituent (s) and a heterocyclic residue optionally having substituent (s); R4, R5, R6, R7, R8 and R9; Each is at least one substituent selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, and an electron withdrawing group, and R4 To R8, two adjacent substituents may be bonded to each other to form a ring. A phosphonic acid diester derivative represented by

3. General formula (1)
(Wherein R1 is OR10, R2 is OR11, and R10 and R11 exclude a hydrogen atom).

  4). The phosphonic acid diester derivative shown in 3 above, wherein R10 is any one of an alkyl group which may have a substituent and an aryl group which may have a substituent, and R11 has a substituent. A phosphonic acid diester derivative which is an aryl group which may have.

  5. The phosphonic acid diester derivative shown in 4 above, wherein the substituent of the alkyl group which may have the substituent is a phenyl group which may have a substituent, a heterocyclic residue having nitrogen, an alkyl A phosphonic acid diester derivative which is at least one group selected from the group consisting of sulfanyl group, arylsulfanyl group, hydroxy group, carbamoyl group, amino group, guanidino group, alkoxy group, amide group, carboxy group and carboxy group equivalent .

  6). The phosphonic acid diester derivative shown in 4 above, wherein the alkyl group which may have the substituent of R10 is represented by the general formula (7)

(Wherein R12 represents any of an alkyl group which may have a substituent, an aryl group which may have a substituent, and a hydrogen atom, and R13 represents a hydrogen atom and a general formula (8)

(Wherein n1 is an integer of 0 to 4, n2 is any of 0 and 1, n3 is an integer of 0 to 4, X1 is either an amide group or an alkenyl group, X2 is a carboxy group, and Any one of the equivalents of a carboxy group represents any one of R14 represents either a hydrogen atom or a lower alkyl group. A phosphonic acid diester derivative.

7). A phosphonic acid diester derivative represented by the above 5 or 6, equivalent of carboxyl group and carboxy _{groups, -COOR, -CONR 2, -COR,} -CN, -NO 2, -NHCOR, -OR, -SR, A phosphonic acid diester derivative, which is at least one group selected from the group consisting of —OCOR, —SO ₃ R, and —SO ₂ NR ₂ , wherein R is any one of a hydrogen atom and an alkyl group.

  8). The phosphonic acid diester derivative shown in 6 above, wherein the substituent of the alkyl group which may have a substituent of R12 is a phenyl group which may have a substituent, or a heterocyclic residue having nitrogen A phosphonic acid diester derivative which is at least one group selected from the group consisting of an alkylsulfanyl group, an arylsulfanyl group, a hydroxy group, a carboxy group, a carbamoyl group, an amino group, a guanidino group, an alkoxy group, and an amide group.

  9. The phosphonic acid diester derivative shown in 4 above, wherein the substituent of the aryl group which may have the substituent may be an alkyl group which may be substituted with either a carboxy group or an equivalent of a carboxy group, A phosphonic acid diester derivative which is at least one group selected from the group consisting of an electron withdrawing group, a carboxy group, and an equivalent of a carboxy group.

  10. The phosphonic acid diester derivative shown in 4 above, wherein the aryl group which may have a substituent is a phenyl group which may have a substituent.

  11. The phosphonic acid diester derivative shown in 10 above, wherein the phenyl group which may have the above substituent is represented by the general formula (9)

Wherein Y1 represents one group selected from the group consisting of —R ′, —OR ′, and an electron withdrawing group, and Y2 is substituted with either a carboxy group or an equivalent of a carboxy group And represents one group selected from the group consisting of an alkyl group optionally having a double bond, a hydrogen atom, a carboxy group, and an equivalent of a carboxy group, and two adjacent substituents Y1 And Y2 may be bonded to each other to form a ring, and R ′ represents either a hydrogen atom or an alkyl group optionally having a double bond.)

12 A phosphonic acid diester derivative represented by the above 9 or 11, an electron withdrawing group, a halogen _{atom, -COOR ', - CONR' 2} , -COR ', - OCOR', - CF 3, -CN, -SR ', At least one group selected from the group consisting of —S (O) R ′, —SO ₂ R ′, —SO ₂ NR ′ ₂ , —PO (OR ′) ₂ , and —NO ₂ , wherein R ′ is A phosphonic acid diester derivative having the same meaning as described above.

13. A phosphonic acid diester derivative represented by the above 9 or 11, equivalent of carboxyl group and carboxy _{group, -COOR ", - CONR" 2} , -COR ", - CN, -NO 2, -NHCOR", - OR _{", -SR", - OCOR "} , - SO 3 R", - " is at least one group selected from ₂ made from a group, R" SO 2 NR and is not a hydrogen atom and a double bond A phosphonic acid diester derivative which is any of the possible alkyl groups.

  14 General formula (10)

(Wherein R12, R14, X2, Y1 and n3 represent the same meaning as described above).

  15. Formula (11)

(Wherein R12, R14, X2, Y1, n1, and n3 represent the same meaning as described above).

  16. Formula (12)

(In the formula, R15 represents a lower alkyl group, and W represents the general formula (13) to the general formula (16).

R16 represents any one of a hydrogen atom and a lower alkyl group, and Y1 and Y2 represent the same meaning as described above. A phosphonic acid diester derivative represented by

  17. Formula (17)

(Wherein R12 and Y1 represent the same meaning as described above).

  18. General formula (18)

(Wherein Y1 and Y2 represent the same meaning as described above).

  19. General formula (19)

(Wherein, M represents an alkali metal, and R17 represents an alkoxycarbonyl group including an optionally substituted aromatic hydrocarbon group), a metal of 2-substituted amino-4-phosphonobutanoic acid salt.

  20. General formula (20)

A 2-substituted amino-4-phosphonobutanoic acid ester represented by the formula (wherein R18 represents an aromatic hydrocarbon group which may have a substituent, and R17 represents the same meaning as described above).

  In addition, the detailed manufacturing method and various properties of the phosphonic acid diester derivatives shown in the above 1 to 20 are disclosed in Patent Document 9. In the present invention, it is added that the description of Patent Document 9 can be incorporated as necessary, and the contents of Patent Document 9 can be appropriately used in the present invention. That is, the disclosed content of Patent Document 9 is also a part of the present invention, and correction based on the described content of Patent Document 9 is also possible.

  In particular, in the general formula (12), the phosphonic acid diester derivative in which W is the general formula (13) or (15) is actually synthesized in the Example of Patent Document 9 and GGT inhibitory activity is also confirmed. This is preferable.

  In addition, it has been confirmed by the present inventors that the phosphonic acid diester derivative exhibits GGT activity when the leaving group is eliminated from the phosphate group. That is, GGT activity improves as the leaving group is easily removed. However, since the ease of leaving of the leaving group is related to the stability of the compound, it is not preferable to use a compound having a leaving group that is easily removed. Examples of the compound having an excellent balance between the detachability of the leaving group and the stability include the phosphonic acid diester derivatives of 1 to 20 above. In the general formula (12), R15 represents the general formula (13 ) Or (15) is more preferred, and 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid is particularly preferred.

  The phosphonic acid diester derivative may contain not only a simple substance and a mixture but also an optical isomer. For example, in the case of 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid used in the Examples, since there are two chiral centers, there are a total of four types of optical isomerism. It is a mixture of bodies.

  The skin fibroblast protein production promoter or keratinocyte migration / proliferation promoter with GGT inhibitory activity of the present invention is used to prevent or improve wrinkles and sagging by improving skin tension and glossiness, or for ultraviolet damage ( It can be used by applying to the skin as a composition for dermatology, such as prevention / improvement of photoaging) or maintenance / improvement of skin moisturizing function, and also as a poultice, cosmetics, bath preparation, and sometimes as a subcutaneous injection. The dosage form can be arbitrarily selected according to the purpose. In particular, it can be widely used as a cosmetic, and can be in the form of creams, ointments, emulsions, solutions, gels, powders, granules, etc., and packs, lotions, powders, sticks, etc. . The preparation forms are also aqueous, solubilized, emulsified, powder, oil / liquid, gel, ointment, aerosol, water-oil two-phase, water-oil-powder three-phase, etc. can do. That is, if it is basic cosmetics, it can be widely used in the above-mentioned various dosage forms in the form of face wash, lotion, milky lotion, cream, gel, essence (beauty serum), pack, mask and the like. In addition, makeup cosmetics can be widely used in the form of foundations and toiletry products such as body soaps and soaps. Furthermore, as a pharmaceutical etc., it can utilize widely in forms, such as various ointments and creams. Further, these dosage forms and forms are not limited to the preparation forms of the skin fibroblast protein production promoter and the keratinocyte migration / proliferation promoter by the GGT inhibitory activity of the present invention.

  The blending amount of the GGT inhibitor in the protein production promoter for dermal fibroblasts or the keratinocyte migration / proliferation promoter of the present invention is not particularly limited, and the type, properties, quality, and expected effect of the formulation to be blended For example, it is 0.00001 to 50.0% by weight, more preferably 0.00005 to 10.0% by weight, and even more preferably 0.0005 to 1.0% by weight. In particular, when penetration is promoted by, for example, making a GGT inhibitor into a liposome, the effect can be expected even with a smaller amount.

  In one embodiment, when the skin fibroblast protein production promoter or keratinocyte migration / proliferation promoter of the present invention is applied to skin fibroblasts in vitro, the protein production of the skin fibroblasts of the present invention is as follows. The compounding amount of the GGT inhibitor in the promoter or the keratinocyte migration / proliferation promoter is 0.00033% by weight. In another embodiment, when the dermal fibroblast protein production promoter or keratinocyte migration / proliferation promoter of the present invention is applied to human skin in vivo, the protein production of the dermal fibroblasts of the present invention is as follows. The compounding amount of the GGT inhibitor in the promoter or keratinocyte migration / proliferation promoter is 0.00053% by weight to 0.0053% by weight.

  The protein production promoter and keratinocyte migration / proliferation promoter of the dermal fibroblasts of the present invention include, in addition to GGT inhibitors, components that are usually used in dermatological agents such as cosmetics and pharmaceuticals, for example, whitening agents, Moisturizer, antioxidant, oil component, UV absorber, surfactant, thickener, alcohol, powder component, colorant, aqueous component, water, various skin nutrients and vitamins, etc. can do.

  In addition, edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid and other chelating agents, caffeine, tannin, tranexamic acid and its derivatives, licorice extract, glabrizine, various crude drugs , Drugs such as tocopherol acetate, glycyrrhizic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid and other whitening agents, glucose, fructose, mannose, sucrose, Sugars such as trehalose can also be appropriately blended.

  Further, for example, vegetable oils such as olive oil, avocado oil, palm oil, coconut oil, and hardened castor oil, and animal oils such as beef tallow and lard can be used as the fats and oils. Examples of waxes include beeswax, carnauba wax, whale wax, and lanolin. Examples of the mineral oil include liquid paraffin, petrolatum, paraffin, squalene, squalane and the like. For example, as fatty acids, in addition to natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid, synthetic fatty acids such as isononanoic acid and caproic acid can be used.

  Further, as alcohols, natural alcohols such as ethanol and isopropanol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol, ethylene glycol, polyethylene glycol, 1,3-butylene glycol, glycerin, batyl alcohol, Polyhydric alcohols such as pentaerythritol, sorbitol, mannitol, glucose and sucrose can also be used.

  In addition, esters such as isopropyl myristate, metal soaps such as aluminum stearate, gums and water-soluble polymer compounds such as gum arabic, carrageenan, gelatin, alginic acid and its salt, hyaluronic acid and its salt, chondroitin sulfate, carboxy You may mix | blend methylcellulose, carboxyethylcellulose, etc.

  Surfactants (anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants), vitamins, amino acids, moisturizers, animals or plants, herbal extracts or extracts, microbial culture metabolites, α-Hydroxy acids, inorganic pigments, UV absorbers, astringents, bactericides / disinfectants, hair preparations, fragrances, pigments / colorants, other hormones, sequestering agents, pH adjusters, chelating agents, antiseptics Antifungal agent, refreshing agent, stabilizer, emulsifier, animal / plant protein and its degradation product, animal / plant polysaccharide and its degradation product, animal / plant glycoprotein and its degradation product, blood flow promoter, Anti-inflammatory / antiallergic agents, cell activators, keratolytic agents, wound healing agents, foaming agents, thickeners, oral preparations, deodorants / deodorants, bittering agents, seasonings, enzymes, etc. are also available .

  In the above-described embodiment of the present invention, the protein production promoter for skin fibroblasts is a method for promoting protein production of skin fibroblasts using a GGT inhibitor, or the protein production promotion of skin fibroblasts. Can also be read as using a GGT inhibitor.

  Further, the elastin production promoter for skin fibroblasts is a method of promoting elastin production of skin fibroblasts using a GGT inhibitor, or the use of a GGT inhibitor for promoting elastin production of skin fibroblasts. It can be replaced.

  Furthermore, the HSP47 production promoter for skin fibroblasts is a method of promoting HSP47 production of skin fibroblasts using a GGT inhibitor, or the use of a GGT inhibitor for promoting HSP47 production of skin fibroblasts. It can be replaced.

  Further, an α-SMA production promoter for skin fibroblasts is a method for promoting α-SMA production of skin fibroblasts using a GGT inhibitor, or for promoting α-SMA production of skin fibroblasts. It can also be read as the use of a GGT inhibitor.

  Moreover, the mature collagen production promoter of dermal fibroblasts is a method of promoting mature collagen production of dermal fibroblasts using a GGT inhibitor, or a GGT inhibitor for promoting mature collagen production of dermal fibroblasts It can also be read as using. Furthermore, it can be read as a method of promoting collagen maturation of skin fibroblasts using a GGT inhibitor, or use of a GGT inhibitor for promoting collagen maturation of skin fibroblasts.

  Further, the keratinocyte migration / proliferation promoter is a method for promoting keratinocyte migration and / or proliferation using a GGT inhibitor, or a GGT inhibitor for promoting keratinocyte migration and / or proliferation. It can also be read as using.

  Hereinafter, examples will be shown, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these examples.

[Experimental Example 1: Evaluation of elastin production ability, α-SMA production ability, HSP47 production ability, and mature collagen production ability]
In order to investigate the elastin producing ability improving action, α-SMA producing ability improving action, HSP47 producing ability improving action and mature collagen producing ability improving action of the GGT inhibitor, the following experiment was conducted. The specific experimental method is as follows.

(1) Preparation of sample Human skin-derived fibroblasts (CCD-1059SK, DS Pharma Medical Co., Ltd.) are subcultured in DMEM medium containing 10% FBS (fetal bovine serum), and DMEM medium containing 10% FBS The cell number was adjusted to 1.0 × 10 ⁵ cells / ml and 6.0 × 10 ⁵ cells were seeded on a plastic petri dish (Gleiner Japan Co., Ltd.) having a diameter of 60 mm, and 10 μM GGT inhibitor was used during the main culture. Was added and cultured for 24 hours to prepare cells of the 10 μM GGT inhibitor added group. As a GGT inhibitor, 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid (GGT5a) was used.

  As a control, a group of cells to which no GGT inhibitor was added was prepared. After the completion of the main culture, the cells were collected, and a sample for the control group for use in the Western blotting method was prepared by the same method as the 10 μM GGT inhibitor added group.

<Sample group>
1) Control group 2) 10 μM GGT inhibitor added group.

(2) Measurement of the production amount of elastin For the sample group of 1) to 2) prepared in (1), the cells were collected after the completion of the main culture, and the production amount of elastin was determined using an antibody that recognizes elastin (Monoclonal Antibody to Analysis was performed by Western blotting using Elastin-Ascites, Acris Antibodies GmbH.

  The analysis result of the obtained elastin production amount is shown in FIG. (A) of FIG. 1 is a diagram showing the results of Western blotting, and (b) of FIG. 1 is a graph showing the results of quantitative analysis of the band density of Western blotting.

  The results of Western blotting were quantitatively analyzed for band density by image analysis using Scion Image (manufactured by Scion).

  As a result, as shown in FIG. 1, it was confirmed that by adding the GGT inhibitor GGT5a at a concentration of 10 μM, the elastin production ability was significantly improved as compared with the control.

(3) Measurement of production amount of α-SMA With respect to the sample group of 1) to 2) prepared in (1), after the completion of the main culture, the cells were collected, and the production amount of α-SMA actin was measured using α-SMA. The analysis was performed by Western blotting using an antibody to be recognized (Mouse Anti-Human Smooth Muscle Actin (1A4), Dako A / S).

  The analysis result of the obtained amount of α-smooth muscle actin production is shown in FIG. (A) of FIG. 2 is a figure showing the result of a Western blot, and (b) of FIG. 2 is a graph which shows the result of having analyzed the density | concentration of the band of a Western blot quantitatively.

  The results of Western blotting were quantitatively analyzed by the same method as described in the above section “(2) Measurement of production amount of elastin”.

  As a result, as shown in FIG. 2, it was confirmed that the addition of the GGT inhibitor GGT5a having a concentration of 10 μM exhibited a significant α-SMA production ability improving effect as compared with the control.

(4) Measurement of production amount of HSP47 Regarding the sample group of 1) to 2) prepared in (1), after the main culture is completed, the cells are collected, and the production amount of HSP47 is changed to an antibody that recognizes HSP47 (Anti-HSP47 Mouse Monoclonal, Stressgen BIOREAGENTS.CORP) were analyzed by Western blotting.

  The analysis result of the obtained HSP47 production amount is shown in FIG. (A) of FIG. 3 is a figure showing the result of a Western blot, and (b) of FIG. 3 is a graph which shows the result of having analyzed the density | concentration of the band of a Western blot quantitatively.

  The results of Western blotting were quantitatively analyzed by the same method as described in the above section “(2) Measurement of production amount of elastin”.

  As a result, as shown in FIG. 3, it was confirmed that by adding the GGT inhibitor GGT5a at a concentration of 10 μM, the HSP47 production ability was significantly improved as compared with the control.

(5) Measurement of amount of mature collagen For the sample group of 1) to 2) prepared in (1), after the completion of the main culture, the cells are collected, and the amount of type I collagen produced is determined based on an antibody that recognizes type I collagen ( The analysis was performed by Western blotting using COL1A1 (C-18), SANTA CRUZ BIOTECHNOLOGY, INC. The antibody is an antibody that recognizes both mature collagen and precursor collagen, and mature collagen and precursor collagen are detected as proteins having different molecular weights. The amount of mature collagen can be measured by quantitatively analyzing the density of a band having a molecular weight of 70 to 90 kDa representing mature collagen.

  The analysis result of the amount of mature collagen obtained is shown in FIG. (A) of FIG. 5 is a figure showing the result of a western blot, and (b) of FIG. 5 is a graph which shows the result of having analyzed the density | concentration of the band of a western blot quantitatively.

  The results of Western blotting were quantitatively analyzed by the same method as described in the above section “(2) Measurement of production amount of elastin”.

  As a result, as shown in FIG. 5, it was confirmed that by adding the GGT inhibitor GGT5a at a concentration of 10 μM, a remarkable effect of improving the ability to produce mature collagen was obtained compared to the control. In other words, it can be said that the addition of the GGT inhibitor GGT5a at a concentration of 10 μM exhibits a remarkable collagen maturation promoting effect.

  From the above results, it was confirmed that the GGT inhibitor GGT5a can significantly enhance the production amounts of elastin, α-SMA, HSP47 and mature collagen in human skin-derived fibroblasts. In addition, even when the GGT inhibitor GGT5a was added, the number of skin fibroblasts was hardly increased (not shown). This suggests that the GGT inhibitor increased the number of dermal fibroblasts, but did not increase overall protein production, but GGT inhibitors promoted protein production of individual dermal fibroblasts. As a result, it was revealed that the overall protein production increased.

(Discussion)
Skin fibroblasts present in the dermis of the skin produce extracellular matrices such as collagen and elastin. It is known that elastin entangles with collagen and gives skin elasticity. For this reason, it is considered that the enhancement of collagen and elastin production ability of skin fibroblasts is important for maintaining skin tension and preventing wrinkles.

  In Experimental Example 1, the GGT inhibitor contains elastin production in skin fibroblasts, α-SMA production that is an index of skin fibroblast activation, HSP47 production that is involved in collagen maturation, and mature collagen production. It was confirmed that the amount was simultaneously increased.

  As the mechanism of action of the GGT inhibitor GGT5a, the intracellular glutathione concentration is temporarily reduced 6 to 8 hours after the administration of the drug, and thus elastin production, HSP47 production in fibroblasts, It was strongly suggested that α-SMA production and mature collagen production were promoted.

  The mechanism of action of the present GGT inhibitor is completely different from vitamin C, Matrixyl (registered trademark), Matrixyl 3000, etc., which are reported to have an ability to improve collagen production, and are regulated by glutathione response (information) It was strongly suggested that the transmission pathway was working.

  Therefore, in addition to elastin, HSP47, α-SMA and mature collagen, the addition of GGT inhibitor GGT5a induces the expression of a series of cytoprotective proteins and has a mechanism to protect the skin from external stimuli such as ultraviolet rays and dryness. It is thought that there is a possibility that it has been triggered. In other words, the anti-aging effect of GGT inhibitors is considered to have successfully utilized the natural response of cells induced by mild oxidative stress due to a transient weak decrease in glutathione. It is thought to cause a synergistic effect on cytotoxic external stimuli (such as ultraviolet rays) including aging prevention.

[Experimental example 2: Evaluation of migration and proliferation ability of keratinocytes]
(1) Preparation of sample The following experiment was conducted in order to investigate the effect of the GGT inhibitor to improve the migration / proliferation ability of keratinocytes. The specific experimental method is as follows.

Human keratinocytes (human keratinocytes) (HaCat cells, distributed from Osaka City University School of Medicine Dermatology) are seeded in 2.25 × 10 ⁵ plastic dishes (Grineer Japan, Inc.) having a diameter of 35 mm. Culturing until confluent in DMEM medium containing% FBS, then scraping the cells linearly with a pipette tip, adding 1 μM GGT inhibitor or 10 μM GGT inhibitor and culturing for 24 hours, 1 μM GGT inhibitor Samples of the addition group and 10 μM GGT inhibitor addition group were prepared. As a GGT inhibitor, 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid (GGT5a) was used.

  Moreover, the sample of the group which does not add a GGT inhibitor as control was prepared.

<Sample group>
1) Control group 2) 1 μM GGT inhibitor added group 3) 10 μM GGT inhibitor added group

(2) Measurement of migration / proliferation ability As an index for evaluating the migration / proliferation ability of keratinocytes, the degree of repair of the scratch site was measured for the sample groups 1) to 3) prepared in (1). Specifically, the scratch width immediately after scratching (after 0 hours) was measured on the monitor screen to be a (cm), and after 24 hours, the scratch width at the same location was measured to be b (cm).

Next, for the sample groups 1) to 3), the repair width (cm) was determined using the following formula (1).
Repair width = (scratch width a after 0 hours) − (scratch width b after 24 hours) (1)
The repair rate was determined as a relative value when the repair width in the control group was 100%.

  The results are shown in FIG. FIG. 4 is a graph showing the influence of a GGT inhibitor on the promotion of migration / proliferation ability of human keratinocytes. The symbol “a” shown in FIG. 4 represents the scratch width immediately after scratching (after 0 hours), and the symbol “b” represents the scratch width 24 hours after scratching.

  Table 1 shows “repair width (ab) (cm)” and “repair rate (%)” of the sample groups 1) to 3).

  As a result, as shown in FIG. 4 and Table 1, it was confirmed that by adding the GGT inhibitor GGT5a, there was a remarkable effect of improving the scratch repair rate compared to the control group. As the repair rate value is larger, the keratinocyte migration / proliferation ability is enhanced. From the results shown in FIG. 4 and Table 1, the GGT inhibitor GGT5a was added to compare with the control. As a result, it was confirmed that the effect of improving the migration and proliferation ability of keratinocytes was remarkable.

  Moreover, as shown in FIG. 4 and Table 1, the 10 μM GGT inhibitor added group GGT had a larger repair rate value than the 1 μM GGT inhibitor added group. From this result, it was confirmed that the GGT inhibitor can remarkably enhance the migration / proliferation ability of keratinocytes in a concentration-dependent manner.

(Discussion)
The keratinocytes that form the epidermis of the skin divide and proliferate in the basal layer, then move to the horny layer, and eventually become peeled off. For this reason, the enhanced migration and proliferation ability of keratinocytes has an important role in promoting skin metabolism and maintaining the skin barrier function. Experimental Example 2 confirms that the GGT inhibitor enhances the migration / proliferation ability of keratinocytes, and suggests that the GGT inhibitor has the effect of promoting skin metabolism and maintaining the skin barrier function. .

  As described above, the GGT inhibitor has both an effect of promoting the migration / proliferation of keratinocytes in the epidermis and an action of activating skin fibroblasts (promoting production of proteins such as elastin and HSP47). confirmed. Since the skin fibroblast protein production promoter and keratinocyte migration / proliferation promoter of the present invention contain a GGT inhibitor as an active ingredient, the skin aging preventive effect or improvement of skin symptoms by various functions It is thought that it can be used as a new type of drug that exerts an effect.

[Prescription example: serum]
A cosmetic liquid having the following formulation was produced by a conventional method.
(Composition) (wt%)
Sorbit 4.0
Glycerin 3.0
Butylene glycol 3.0
Polyethylene glycol 1500 5.0
POE (20) oleyl alcohol ether 0.5
Sucrose fatty acid ester 0.2
Methylcellulose 0.2
GGT inhibitor 0.005
Purified water Amount of 100 [Prescription example: Emulsion]
An emulsion having the following formulation was produced by a conventional method.
(Prescription) (wt%)
Glycerin 1.5
Sucrose fatty acid ester 1.5
Sorbitan monostearate 1.0
Squalane 7.5
Dipropylene glycol 5.0
GGT inhibitor 0.005
Purified water Total amount of 100 [Prescription example: Cream]
A cream having the following formulation was produced by a conventional method.
(Prescription) (wt%)
Glycerin 3.0
Butylene glycol 3.0
Squalane 19.0
Stearic acid 5.0
Glycerol monostearate 5.0
Sorbitan monostearate 12.0
Polyethylene sorbitan monostearate 38.0
Sodium edetate 0.03
GGT inhibitor 0.005
Purified water Amount of 100 in total.

  The GGT inhibitor has an activity to enhance the production of elastin and HSP47 protein effective in reducing aging of the skin and damage caused by ultraviolet light, and at the same time, an activity to promote the growth of keratinocytes. Have For this reason, the protein production promoter for skin fibroblasts of the present invention or the keratinocyte migration / proliferation promoter of the present invention containing a GGT inhibitor as an active ingredient prevents skin wrinkles and sagging and is fresh. It can be a dermatological agent having a high anti-aging effect for keeping the skin or an improvement effect on skin symptoms.

  Specifically, the present invention is a medicated cosmetic product having anti-aging effects such as reduction of wrinkles on the face, skin tension, and increase in freshness; skin care products such as rough hands, cracks, cracks, frost burns, dry skin in winter, etc. Is available.

  In addition, since the keratinocyte migration / proliferation promoter of the present invention has an action of promoting keratinocyte migration and / or proliferation, it can be a dermatological agent having a high wound healing effect. Specifically, the present invention can also be used as a therapeutic agent (ointment, cream) or the like for pressure ulcers (bed sores).

Claims (2)

2-Amino-4 - {[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid as an active ingredient containing keratinocytes migration and proliferation promoting agent, a wound healing agent. A therapeutic agent for pressure ulcers (bed sores) containing a keratinocyte migration / proliferation promoter comprising 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid as an active ingredient .